A quantitative XANES analysis of the calcium high-affinity binding site of the purple membrane.

In this article we report x-ray absorption measurements of Ca(2+)-substituted bacteriorhodopsin. We present a detailed study of the absorption spectrum close to the absorption edge that is very sensitive to the site geometry. We combined ab initio calculations of the x-ray absorption cross section based on a full multiple scattering approach, with a best fit of the experimental data performed by changing the cluster geometry. The Ca(2+)-bacteriorhodopsin environment is composed of six oxygen atoms showing a distorted orthorhombic symmetry, whereas the Ca(2+) in water solution has a regular octahydrated first sphere of coordination. Our results are in good agreement with previous molecular models suggesting that the high-affinity cationic site could be in the proximity of the retinal pocket. Our results provide strong direct evidence of the specific binding site of the metal cation in bacteriorhodopsin.